Brief write-up about work done & future directions

Implantation of the embryo is an extremely complex process but this process along with placentation is the initial requirement for the propagation of a species. These two processes provide the initial platform for protecting & nourishing their offspring. The successful continuance of a species is thus very much dependent on this process & involves cross-talk between the maternal genome, the embryo & the placenta - all directed to the embryo’s advantage. Failures in embryo implantation & placental development are clinically important since one-third of normal human pregnancies end in spontaneous abortion out of which 75% represent a failure of implantation & this is also a major limiting factor in assisted reproduction.

My groups work has been directed in unraveling the molecular mechanisms mediating embryo implantation along several roads to reach the summit of implantation.

ROS & female reproduction (DST sponsored grants):

1. ROS in uterus: Redox signaling involving ROS generation at embryo Implantation:
   Our group is the pioneer labwhich showed that superoxide radical is required in a normal physiological process. We have established the involvement of reactive oxygen species under an estrogen switch for successful embryo implantation. Our attempts to identify the superoxide generating machinery in the uterus have narrowed down onto NADPH oxidase. We have identified a PHOX homolog (NADPH oxidase, NOX) in mouse uterus that shows activation during the window of implantation and this NOX is an atypical multimeric enzyme system. This NOX assembly is triggered by estrogen invoking the non-genomic Membrane Initiated Steroid Signaling Pathway.






2. ROS & embryo:
   

   Immense superoxide generated by peri-implanting embryos triggers "zona hatching" and this is critical for embryo implantation.

   
   

   On the ground of these results, free-radical scavengers have been successfully used to inhibit implantation in mice when delivered intraluminally prior to implantation. Superoxide quenchers abrogate embryo hatching and implantation thus opening up a new vista to female fertility control without playing with the normal hormonal/immune system. Currently we are deciphering the superoxide generator in embryos (ICMR sponsored).





3. Membrane modifications via superoxide radical
   

   Deliberate & controlled superoxide generation leads to membrane fluidity enhancement and not rigidification as widely believed by formation of intermediate kinked species. The endometrial membrane at embryo implantation is highly polar and fluid due to existence of dienyl radicals. This predicts its role in modulating endometrial membrane architecture enabling successful implantation.

Mechanism of immune tolerance at embryo implantation

Pregnancy is a state synonymous to a controlled and regulated cancerous invasion of the blastocyst which is a semiallograft and its subsequent growth in the background of a seamless network of factors where it has to evade rejection by a cohort of female immune responses. The survival of an embryo bearing the paternal antigens within the immunocompetent environment of the maternal uterus renders 'pregnancy' to be a state of immunological paradox. The ratio of Th1/Th2 responses are crucial for pregnancy maintenance. Monocyte Chemotactic Protein-3 is a pro-inflammatory, CC chemokine and a Th1 effector which is capable of eliciting significant anti-tumoral immune responses. A significant MCP3 down-regulation at the “implantation window” in the uterus and the implanting embryo is imperative to a successful pregnancy. Injection of Ru486, a progesterone antagonist, leads to a massive surge in MCP3 expression in both immature mice and delayed implantation models. The cross-talk between Ru486 and amplified MCP3 expression may be one of the mechanisms by way of which RU486 performs its abortificient and anti tumor role.

Genomics & Proteomics of Embryo Implantation:

Implantation-associated gene/protein expression in mammalian uterus. (sponsored by the Rockefeller Foundation/CONRAD)

1. Identification & characterization of a transient 32.6 kDa - protein:
   The idea that implantation of the mammalian embryo could be mediated by specific macromolecule(s) expressed over the surfaces of the interacting trophoblast and uterine epithelia has been the major focus of my recent research. We have identified a short-lived novel 32.6kD protein in the mouse uteri expressed precisely at the time of implantation (5.00 a.m. on day 5.00 of pregnancy).This protein appears to be very interesting in that its "transient" expression could suggest its function as a "receptor" for the blastocyst in the uterus. The gene is successfully sequenced and deposited in GENEBANK. The expression of this protein/gene at Implantation & under the control of Progesterone. RU 486 - a progesterone antagonist inhibits its expression. Our recent homology modeling studies predict this protein as a transcription factor of the “winged helix class”. Recently, we have deciphered the DNA binding sequence for this protein. We are truing to assigning a functional role for ‘Implantin’ using molecular modeling & wet-lab experiments.


2. Proteomics at embryo implantation:
   We have identified a couple of protein candidates including NOS, CrkL, STATs, I -121 & D260 using a one-by-one approach which could play crucial role in establishing a receptive environment for blastocyst adhesion. We have also short-listed a 121 kDa protein and a D260 protein to be critical for embryo implantation. We are trying to characterize and assessing the contraceptive potential of these two protein candidates.
   Our overall goal is to expression profile of proteins expressed at the receptive phase of uterus so as to generate signature patterns that reflect the competence of uterus.


3. Identifying interacting partners of estrogen receptor using a proteomic approach (CSIR sponsored) :
   In order to pin-point the role of estrogen receptor in triggering events leading to embryo implantation we are in the process of identifying interacting partners of estrogen receptor using a MALDI-TOF peptide finger-printing of proteins.

Signalling mechanism rife at embryo implantation (sponsored by BRNS & CSIR)

Embryo implantation is an event which involves close interaction between the mother & the embryo. The cross-talk between the two is mediated by a battery of signaling cascades emanating from both the sides so as to finally culminate in a successful implantation. We have recently elucidated the tranlocation of crkL to the nucleus at the "window of implantation” and this process could initiate signaling cascades.
Since the process of implantation requires immense extra-cellular matrix signaling and cytoskeletal reorganizations, we try to analyze these changes critically.

Embryo development & acquisition of stem cell characteristics (CSIR/ICMR sponsored)

The activation of the embryo is crucial prior to implantation. Efforts are directed to identifying intrinsic factors responsible for embryo activation with special focus on transcription factors.

Pathogenesis of disease

We undertake research to understand the causes of female infertility on one hand while on the other hand we are keen on studying signaling defects during the development of pathogenesis of diabetes.